IGF-II analogues

ABSTRACT

Insulin-like Growth Factor II (IGF-II) analogues in which at least one of R37 and R38 is replaced with another amino add residue, the most preferred being IGF-II R37Q R38Q, can readily be produced in E. coli, unlike natural IGF-II, which is cleaved on secretion. The analogues retain activity on the type I and type II IGF receptors but have lower affinity for the insulin receptor; they are therefore more specific in their action.

This invention relates to analogues of human insulin-like growth factorII (IGF-II) and to nucleic acid coding for them.

Human IGF-II belongs to a family of growth factors that includesinsulin, relaxin, and insulin-like growth factor I (IGF-I). Members ofthe family share limited sequence homology but are presumed to exhibitsimilar structures on the basis of a conserved pattern of disulphidebond formation (Dafgard et al, Journal of Cell Science, 3:53-64 (1984).IGF-I or somatomedin C is a mitogen that mediates the growth-stimulatoryeffects of growth hormone throughout childhood and adolescence. The roleof the related protein IGF-II is more obscure though it has beenimplicated in regulating brain and muscle development, placental growthand the stimulation of bone and cartilage formation. IGF-II is found athighest levels in bone (1,750 ng/g dry wt.), about a ten fold higherconcentration than for IGF-I (190 ng/g dry wt.). Studies involving insitu hybridisation have revealed that both IGF-I and IGF-II mRNA areproduced predominantly in cells of mesenchymal origin. This suggeststhat both IGFs may be involved in paracrine action on multiple celltypes throughout development with each IGF having its own spectrum oftargets.

The mature form of IGF-II is 67 amino acids in length, its sequencefirst elucidated by Riner et al, FEBS Letters, 89:293 (1978). The maturemolecule is derived from a precursor which includes a 19 amino acidsignal peptide and an 89 amino acid C-terminal extension. In addition, avariant form of IGF-II has been described that possesses an extra 3amino acids in the mature protein. The primary structure of IGF-II isshown in FIG. 1 (SEQ ID NO. 1).

The effects of IGF-II on cells are mediated by at least three differentreceptors. It has a high affinity for the type I IGF receptor, amembrane bound tyrosine kinase with similar organisation to the insulinreceptor. Along with IGF-I, it is likely that the bulk of its effectsare mediated by this receptor. IGF-II also binds with high affinity tothe type II IGF receptor, a membrane bound protein recently identifiedas the lysosomal mannose-6-phosphate acceptor (Tong et al., Journal ofBiological Chemistry, 263:2585-2588 (1987)). Finally, IGF-II can bind tothe insulin receptor, though with lower affinity. It is thought thatthis last interaction is responsible for the hypoglycaemia caused bycertain turnouts that secrete large amounts of IGF-II (Daughaday et al.,New England Journal of Medicine, 319:1434-1440 (1988).

A range of pharmacological effects have been demonstrated for IGF-II,both from in vitro and in vivo studies. In vitro, IGF-II stimulates theproliferation of pre-osteoblasts, inhibits the proliferation of matureosteoblasts and stimulates collagen production by mature osteoblasts. Invivo, by osmotic infusion in the rat at 10 μg/day, IGF-II has been foundto increase serum/bone alkaline phosphatase levels, increase tibialperiosteal bone formation and bone apposition rate, and increase thevertebral forming surface.

The biological effects of both IGF-I and IGF-II are modulated by anumber of binding proteins--see Baxter, R. C., Comparative Biochemistryand Physiology, 91B:229-235 (1988). Some of these serve simply ascarrier proteins in the serum and amniotic fluid. Others, which are moretissue specific in their distribution, are inhibitory and probablyfunction as autocrine or paracrine regulators of cell growth.

The following patent publications cover various aspects of IGF-II:

1. GB-A-0216375 (Amano Pharmaceutical Co. Ltd.) discloses the productionof IGF-II by culturing pituitary cells from a patient with acromegaly.

2. EP-A-0135094 (Amgen) discloses the amino add sequence of IGF-I andIGF-II and nucleotide sequences of genes encoding them. Varioushypotheses for their utility are put forward but nothing concrete by wayof clinically useful activity is proposed, at least for IGF-II, otherthan the treatment of pituitary dwarfism.

3. WO-A-8600619 and EP-A-0189481 (Chiron Corporation) discloseprepro-IGF-I and prepro-IGF-II, but again gives no clinical utility forthe end peptides.

4. EP-A-0193112 (Columbia University) discloses eDNA encoding IGF-II.Again no specific clinical utility is disclosed or forecast for IGF-II.

5. EP-A-0280460 (Eli Lilly) discloses the use of IGF-II in topical woundhealing compositions at a dose which does not produce a systemicinsulin-like effect (i.e. does not effect serum glucose levels).

6. EP-A-0128733 (Genentech) discloses the production of `various forms`of human IGF and EGF by recombinant DNA technology. There is no specificdisclosure of clinical utility beyond saying that human IGF can be usedas a human growth factor.

7. WO-A-8905822 and EP-A-346429 (GroPep) disclose peptide analogues ofIGF-I or IGF-II in which at least the glutamic acid residue is absent atposition 3 from the N-terminal of IGF-I or at position 5 or 6 from theN-terminal of IGF-II.

8. WO-A-9015142 (GroPep) discloses the production of IGF-I or IGF-IIfusion proteins using suitable expression vectors.

9. EP-A-0230869 (Kabigen AB) discloses the use of eDNA coding forimmunoglobulin-G binding domains which are useful in the production oftransformed hosts for production of foreign proteins such as IGF-I andIGF-II.

10. EP-A-0361956 (Eli Lilly) discloses DNA sequences encoding forprotein derivatives such as IGF-I, IGF-II which can be produced in anatural form without non-natural amino acids.

11. EP-A-0224885 (Wakunaga Seiyaku Kabushika Kaisha) discloses the useof growth factors (EG, IGF-II) for enhancing antitumour actions ofantitumour agents or treatments including those against which tumour orcancer has acquired resistance or reduces side effects due to theantitumour agents or treatments.

A number of approaches to the production of both IGF-I and IGF-II havebeen described. IGFs were originally purified from human serum, andIGF-II has been produced by cell culture. The potential advantages ofusing a recombinant DNA approach led a number of groups to investigatethe expression of IGFs in E. coli and yeast. Production of recombinantIGF-I was successfully demonstrated in yeast by secretion as a fusionwith the leader and pro-sequences from the S. cerevisiae α-factor matinghormone (see for example Shuster et al., Gene, 83:47-55 (1989)). Highlevel production of IGF-I from E. coli was achieved by employing aprotein A/IGF-I fusion which directed the secretion of the fused productinto the periplasmic space (Moks et al., Bio/Technology, 5:379-382(1987)). The fusion protein could be isolated by virtue of the affinityof the protein A tail for an immunoglobulin column, and the mature IGF-Ireleased by treatment with hydroxylamine. Both of these methods giverise to correctly folded material. Both methods, however, are associatedwith problems. The yeast derived material is subject to variable amountsof O-linked glycosylation (Hard et al., FEBS Letters, 248:111-114(1989), and the E. coli-derived material contains a number ofcontaminants including proteolytically nicked, oxidised and norleucinecontaining forms (Forsberg et al., Biochemical Journal, 271:357-363(1990).

Production of recombinant IGF-II has proved more problematic. Itssuccessful production in yeast has not been described. Our ownexperience, using a similar approach to that described above for IGF-I,is that only very low level secretion can be obtained, probably due toproteolysis in the ER or golgi apparatus. Similarly, secretion from E.coli results in far lower levels of product than can be obtained withIGF-I. This again has been shown to be due to proteolysis, primarilywithin the C region of the molecule (Hammarberg et al., PNAS,86:4367-4371 (1989)). Full length IGF-II can be obtained by secretionfrom E. coli if a dual fusion approach is employed (ibid). This involvesan N-terminal protein A fusion, to direct secretion of the material, anda C-terminal fusion to a region of the Staphylococcal protein G receptorthat mediates binding to human serum albumin (HSA). This C-terminalfusion provides some protection against proteolysis and allows forselective purification of full-length material by passage down a columncarrying immobilised HSA. This strategy is elegant but complex, andrequires additional steps to cleave the IGF-II from the fusion tails.Furthermore, the use of CNBr to cleave the C-terminal tail leaves amodified C-terminus carrying homoserine or homoserine lactone. Anothersuccessful strategy has been to produce IGF-II intracellularly as afusion protein carrying 45 amino acids from the trp leader and trpEpolypeptide (Furman et al., Bio/Technology, 5:1047-1051 (1987)). Thehigh level expression of this protein directed by a trp promoterconstruct leads to its accumulation as inclusion bodies. This affordsprotection against proteases and simplifies the initial steps of thepurification. The authentic material can be obtained after CNBr cleavageof the fusion protein and refolding under controlled conditions to allowfor the correct formation of disulphide bonds. None of these approachesto the production of IGF-II is ideal, however, because of the problemsassociated with proteolysis. In particular, the complexities introducedby the necessity for chemical cleavage and/or refolding steps makes themless than suitable as production processes. An ideal approach would bethe direct production of correctly folded material by high levelsecretion from a widely used host such as E. coli.

There is much interest in the use of IGFs in the treatment of growthdisorders, osteoporosis and other osteopaenias, muscle wasting diseasesand wound healing. It is therefore of considerable utility to producenew forms of IGF with improved production characteristics and enhancedbiological properties. In particular the sensitivity of IGF-II toproteases and its tendency to induce insulin-like effects at high dosesare real obstacles to its therapeutic use.

The invention seeks to solve the problems of providing an improvedmethod for producing IGF-II and of providing engineered forms of IGF-IIwith improved pharmacological properties. It has been discovered thatcertain mutants in the C region of IGF-II were no longer cleaved onsecretion from E. coli, and that whilst they retained activity, and atleast in some cases full activity, on the type I and type II IGFreceptors, they exhibited lower, at least in some instances 10-foldlower, affinity for the insulin receptor. This was a surprising result,not predicted from modelling studies.

According to a first aspect of the invention, there is provided anInsulin-like Growth Factor II (IGF-II) analogue in which at least one ofR37 and R38, wherein R37 represents the natural arginine residue atposition 37 and R38 represents the natural arginine residue at position38, is replaced with another amino acid residue.

Preferably, both R37 and R38 are replaced. If they are both replaced,they need not be replaced with the same amino acid residue, although itmay be preferred for both replacement amino acid residues to be the samein at least some circumstances.

It is preferred that the or each replacement amino acid residue benon-basic. Non-basic residues include:

    ______________________________________                                        Alanine          Ala         A                                                Valine           Val         V                                                Leucine          Leu         L                                                Isoleucine       Ile         I                                                Proline          Pro         P                                                Phenylalanine    Phe         F                                                Tryptophan       Trp         W                                                Methionine       Met         M                                                Glycine          Gly         G                                                Serine           Ser         S                                                Threonine        Thr         T                                                Cysteine         Cys         C                                                Tyrosine         Tyr         Y                                                Asparagine       Asn         N                                                Glutamine        Gln         Q                                                Aspartic acid    Asp         D and                                            Glutamic acid    Glu         E.                                               ______________________________________                                    

Basic residues apart from arginine (Arg, R) include lysine (Lys, K) andHistidine (His, H).

Preferred non-basic residues are the neutral residues (i.e. those otherthan Asp and Glu) and those other than Pro. The amide residues (Asn andGln, particularly Gln) are the most preferred. IGF-II with themodifications Arg37Gln and Arg38Gln (SEQ ID NO.2) is a good example ofthis aspect of the invention.

Other particular examples include:

    ______________________________________                                        Arg 37              Arg 38 Gln                                                Arg 37              Arg 38 His                                                Arg 37              Arg 38 Pro                                                Arg 37 Pro          Arg 38                                                    Arg 37              Arg 38 Ala                                                Arg 37 Ser          Arg 38                                                    ______________________________________                                    

Apart from the above modifications, a limited number of othermodifications may be made to the natural IGF-II sequence, provided thatthe required biological activity is not lost. For example, up to fivefurther amino acid residues may be modified by way of replacement,addition or deletion, although it is preferred that only one or twoother amino acid residues be modified. Of course, in some circumstancesit may be appropriate to couple an IGF-II analogue to a proteinaceouscarrier, in which case a large number of additional amino acid residues,attributable to the carrier, may be present.

IGF-II analogues in accordance with the invention may in principle bemade by any appropriate means. In general, therefore, according to asecond aspect of the invention, there is provided a process forpreparing an IGF-II analogue as described above, the process comprisingcoupling together successive amino acid residues and/or ligating oligo-and/or poly-peptides. Most conveniently, recombinant DNA technology isused to prepare the IGF-II analogues in an appropriate host cell; inthese circumstances, successive amino acid residues will be coupledtogether ribosomally under the direction of nucleic acid, which alsoforms part of the invention.

According to a third aspect of the invention, there is provided nucleicacid coding for an IGF-II analogue as described above. The nucleic acidwill usually be DNA, but RNA is also within the scope of the invention.DNA in accordance with this aspect of the invention will usually besynthetic or recombinant and may, but will not necessarily, be inisolated form.

Recombinant DNA in accordance with the invention may be in the form of avector. The vector may for example be a plasmid, cosmid or phage.Vectors will frequently include one or more selectable markers to enableselection of cells transfected (or transformed: the terms are usedinterchangeably in this specification) with them and, preferably, toenable selection of cells harbouring vectors incorporating heterologousDNA. Appropriate start and stop signals will generally be present.Additionally, if the vector is intended for expression, sufficientregulatory sequences to drive expression will be present. Vectors notincluding regulatory sequences are useful as cloning vectors.

Cloning vectors can be introduced into E. coli or another suitable hostwhich facilitate their manipulation. According to another aspect of theinvention, there is therefore provided a host cell transfected ortransformed with DNA as described above.

DNA in accordance with the invention can be prepared by any convenientmethod involving coupling together successive nucleotides, and/orligating oligo- and/or poly-nucleotides, including in vitro processes,but recombinant DNA technology forms the method of choice.

According to a fourth aspect of the invention, there is provided aprocess for the preparation of nucleic acid as described above, theprocess comprising coupling together successive nucleotides and/orligating oligonucleotides. IGF-II analogues in accordance with the firstaspect of the invention may be medically useful. According to a fifthaspect of the invention, there is therefore provided an IGF-II analogueas described above for use in medicine, particularly in the treatment ofgrowth disorders, osteoporosis and/or other osteopaenias, muscle wastingdiseases and/or wound healing.

According to a fifth aspect of the invention, there is provided the useof an IGF-II analogue in accordance with the first aspect in themanufacture of a medicament for use in the treatment of growthdisorders, osteoporosis and/or other osteopaenias, muscle wastingdiseases and/or wound healing. It win be appreciated that the inventioncan be used in a method for the treatment or prophylaxis of growthdisorders, osteoporosis and/or other osteopaenias, muscle wastingdiseases and/or wound healing, the method comprising administering to asubject an effective amount of an IGF-II analogue as described above.

According to a sixth aspect of the invention, there is provided apharmaceutical composition comprising an IGF-II analogue as describedabove and a pharmaceutically acceptable carrier therefore.

Appropriate pharmaceutical compositions and formulations can be preparedby those skilled in the art. Since pharmaceutical compositions inaccordance with the invention contain proteins, they will generally beadministered parenterally, for example by injection (whetherintravenous, subcutaneous or intramuscular) or by implantation. For thisreason compositions of the invention will generally be sterile. Suitablecarriers may include water for injections and phosphate-buffered saline.Dosages will generally be determined by the physician or clinician.

Another aspect of the invention relates to solving the problem of how toproduce natural IGF-II or an analogue, whether as described above ornot, in E. coli. It has been discovered that if the IGF-II or analogueis expressed in E. coli as a fusion protein with at least a part ofhorseradish peroxidase (HRP), the difficulties caused by degradation aremuch reduced. HRP is particularly useful because it is expressed at highlevels in E. coli and forms inclusion bodies and because a syntheticgene for HRP has been described (WO-A-8903424) and is commerciallyavailable (British Bio-technology Limited, Laboratory Products Division,Abingdon, Oxfordshire, UK). The fusion protein produced can be cleaved(for example with cyanogen bromide) and the IGF-II or analoguerecovered.

In a seventh aspect, the invention therefore provides a process for theproduction of Insulin-like Growth Factor II (IGF-II), or an analoguethereof, the process comprising culturing Escherichia coli containing anexpressible gene coding for a fusion protein, wherein the fusion proteincomprises at least part of the sequence of horseradish peroxidasecleavably linked to IGF-II or an analogue thereof, under such conditionsas to allow the said gene to be expressed, cleaving the fusion proteinso formed and recovering the IGF-II or analogue.

The part of the sequence of HRP will generally be sufficient forinclusion bodies to be formed. The N-terminal may be included.Preferably, when cyanogen bromide is to be the cleavage agent, the HRPsequence does not contain any methionine residues except adjacent to theIGF-II or analogue sequence, to ensure unique cleavage. The first 53amino acids of HRP have been found to be suitable. Of course, thisarrangement is not essential, as cleavable linkages can easily beengineered between the HRP and IGF-II (or analogue) sequences.

IGF-II analogues which can be produced by means of this aspect of theinvention are those which do not differ in material respects (as far asthis production process is concerned) from natural IGF-II. In general nomore than ten, and preferably no more than five, amino acid changes fromthe natural sequence will be present. Particular IGF-II analogues whichcan be produced by the process of this aspect of the invention includethose of the first aspect.

An eighth aspect of the invention provides an E. coli expression vectorcomprising expressible DNA encoding a fusion protein, wherein the fusionprotein comprises at least part of the sequence of horseradishperoxidase cleavably linked to IGF-II or an analogue thereof. Host cellscontaining such a vector also form part of the invention.

Preferred features of each aspect of the invention are as for each otheraspect, muatatis mutandis.

The invention will now be illustrated by the examples. The examplesrefer to the accompanying drawings, in which:

FIG. 1 shows the primary structure of IGF-II;

FIG. 2 shows the primary structure of IGF-II (Q³⁷ Q³⁸);

FIG. 3 shows a synthetic gene for IGF-II;

FIG. 4 shows the construction of pSD15;

FIG. 5A and 5B shows the strategy used for IGF-II mutagenesis;

FIG. 6A and 6B shows the expression of IGF-II and mutant variants;

FIG. 7A-B show the purification of IGF-II (Q³⁷ Q³⁸);

FIG. 8 shows the construction of plasmid pSD28.

FIG. 9A and 9B shows the purification of IGF-II by the HRP fusion route;

FIG. 10 shows the performance of IGF-II and analogues in accordance withthe invention in a Type 1 IGF receptor binding assay;

FIG. 11 shows the performance of IGF-II and analogues in accordance withthe invention in a Type 2 IGF receptor binding assay; and

FIG. 12 shows the performance of IGF-II and analogues in accordance withthe invention in an insulin receptor binding assay.

EXAMPLES Example 1

Construction of IGF-II Derivatives Carrying Mutations at R37 and R38

The sequence of a synthetic gene for IGF-II (SEQ ID NO. 3) is depictedin FIG. 3. The synthesis of this gene has been described in detail inWO-A-8903423. The synthetic gene was cloned as a NdeI/BamHI fragmentinto the E. coli expression vector pGC517 to give pGC518. In order todirect the secretion of IGF-II, an adapter was synthesised that encodedthe leader sequence for the ompA gene product (SEQ ID NO. 5). Theadapter consisted of two complementary oligonucleotides of 102 and 96base pairs which upon annealing result in the formation of NdeI and SacIcohesive ends. This adapter was cloned between the NdeI and SacI sitesof pGC518 to give the IGF-II secretion vector pSD 15. As secretionvectors are known to be toxic in E. coli, the clone was isolated in thelacI^(q) strain HW1110 to minimise expression in the absence of inducer.The plasmid was re-isolated and the nucleic acid sequence of the ompAand IGF-II regions confirmed. The plasmid was also transformed intoJM103; this strain was deposited on 5th Dec. 1990 at the NationalCollection of Industrial and Marine Bacteria Limited, 23 St. MacharDrive, Aberdeen, AB2 1RY and given accession number NCIMB 40342. FIG. 4details the construction of pSD15.

IGF-II variants carrying mutations at position Arg37 and/or Arg38 wereconstructed by site-directed mutagenesis. An M13mp18 derivative carryingIGF-II was constructed by cloning a Bg/II-SalI fragment encompassingIGF-II and the ompA leader from pSD15 between the BamHI and SalI sitesin the polylinker. Single stranded DNA was isolated and used as atemplate in the mutagenesis procedure. Two primers were used. The first,BB1551 (5'-CCTCTAGATTGCTGAGATACACG-3', SEQ ID NO. 7), was a 23mer thatdirected the substitution of Arg37 and Arg38 with glutamine residues(R37Q, R38Q). The second primer, BB1550(5'-GCCTCTAGANNNNNNNAGATACACG-3', SEQ ID NO. 8), was a 24mer thatdirected the randomisation of the residues Arg37 and Arg38. This wasachieved by incorporating all four nucleotides during the synthesiscycle at the six bases that are complementary to the two argininecodons. Following the mutagenesis, mutant plaques were identified bygrowing small isolates of phage from each, purifying single stranded DNAand dideoxy sequencing using M13 universal primer. With BB1551, a mutantincorporating the desired mutation was identified. With BB1550, 11mutant isolates were obtained carrying various substitutions at Arg36,Arg37 or both positions. The sequence of these mutations is summarisedin Table 1. All mutant clones were plaque-purified and used to infectlarger cultures of JM103. This allowed the isolation of double strandedreplicative form (RF) DNA from each of the mutant isolates.

                  TABLE 1                                                         ______________________________________                                        BB 1550 and 1551 IGFII Mutations                                                                  Mid Selection of Lower                                                                       Amino Acid                                 Constr. No. Primer  Strand Primer  Change                                     ______________________________________                                        pSD41       BB1550  AGA.GTCGTT.AGA gln gln                                    pSD42       BB1551  AGA.GCGGTT.AGA arg gln                                    pSD43       BB1551  AGA.GCGGTA.AGA arg his                                    pSD44       BB1551  AGA.GCTGGA.AGA arg pro                                    pSD45       BB1551  AGA.GGGGCG.AGA pro arg                                    pSD46       BB1551  AGA.GCGCGT.AGA arg ala                                    pSD47       BB1551  AGA.TCGGCG.AGA ser arg                                    pSD48       BB1551  AGA.GATGGC.AGA leu pro                                    pSD49       BB1551  AGA.GCGATA.AGA arg tyr                                    pSD50       BB1551  AGA.GCTFAA.AGA arg leu                                    pSD51       BB1551  AGA.GGGGCT.AGA pro arg                                    pSD52       BB1551  AGA.CGGCAT.AGA ala val                                    BB 1550 sequence                                                                              CGACATAGA.nnnnn.AGATCTCCG                                     BB 1551 sequence                                                                              GCACATAGA.GTCGTT.AGATCTCC                                     ______________________________________                                    

The mutations were introduced into the IGF-II expression vector byisolating them from the mutant RF preparation on a NdeI-SalI fragmentwhich was then cloned the NdeI and SalI sites of pSD 15. The identity ofthe mutations was then re-confirmed by dideoxy sequencing. All themutant derivatives of pSD15 were then transformed into E. coli strainHW1110 (lacI^(q)) for expression studies. The mutagenesis strategy issummarised in FIG. 5.

Example 2

Expression of IGF-II and Mutant Derivatives by Secretion from E. coli

The expression of wild-type IGF-II and its mutant variants was assessedby pulse chase analysis. Single colonies were picked and used toinoculate 5 ml of M9 medium containing 100 μg/ml carbenicillin. Thecultures were incubated at 37° overnight. In the morning, 200 μl of thesaturated cultures was used to inoculate 10 ml of fresh M9 medium, andincubated with shaking until the optical density at 670 nm reached 0.45.A 5 ml aliquot of each culture was then transferred to a fresh universaltube and induced by the addition of 50 μl of 0.5M IPTG. The cultureswere shaken for a further 60 min at 37° . Each culture was thenpulse-labelled by the addition of ³⁵ S-cysteine to a final concentrationof 20 μCi/ml. The cultures were incubated for a further 5 min, afterwhich the labelling was terminated by the addition of 100 μl of coldL-cysteine at 5 mg/ml. The incubation was continued for a further 15min. The cell concentration was determined by measuring the O.D. at 670nm and the cultures were then divided into 1 ml aliquots, the cellscollected by centrifugation at 10,000 rpm for 1 min in a Microfuge(microcentrifuge), the supernatants decanted and the cell pellets frozenon dry ice and stored at -70° C.

The cell pellets were treated to release soluble proteins as follows:The pellets were resuspended in ice-cold resuspension buffer comprising50 mM TRIS pH 8.0, 50 mM NaCl, 0.2 mM PMSF to give a cell concentrationequivalent to 10 absorbance units per ml of cells based on the finalcell concentration determined at the completion of the pulse chase.Lysozyme solution was then added to a final concentration of 0.2 mg/mland the cells incubated on ice for 15 min. The cells were then subjectedto three cycles of freeze-thaw using a dry ice/methanol bath and a waterbath at 25°. The cell lysates were centrifuged at 15,000 g for 30 min topellet the cell debris and any insoluble material. The supernatants weretransferred to a fresh micro-testtube and stored frozen at -20 °.

For SDS PAGE analysis of the cell lysates, 10 μl of each supernatant wasmixed with 5 μl of sample buffer comprising 6M urea, 10% v/v glycerol,5% v/v b-mercaptoethanol, 3% w/v SDS, 60 mM TRIS pH 6.8, 0.01% v/vbromophenol blue. The tubes were tightly sealed and incubated at 100° C.for 5 min. Gel electrophoresis was performed with 1 μl of sample on aPhast gel system (Pharmacia) using commercially available 8-25% Phast(Trade Mark) gradient gels. Pre-labelled MW size standards (Amersham)were also run on each gel. After electrophoresis, the gel was fixed in7% v/v acetic acid, soaked in Amplify (Trade Mark of Amersham), airdried and exposed to X-ray film (Fuji).

The results are depicted in FIGS. 6A and 6B. Cultures carrying theIGF-II expression plasmid pSD15 (wild-type IGF-II) show a band ofbetween 3-4000 Da which is not present in control cultures (uninduced orwithout insert). There is no band present at 6-7000 Da, the expectedsize of IGF-II. In contrast, all the cultures expressing IGF-II mutantderivatives carrying substitutions at either Arg36 or Arg37 or both,exhibited an intense band of the expected molecular weight. The mutantsdid not show a band at the same location as the degraded IGF-IIexpressed from pSD15. Furthermore, a preliminary investigation of thereceptor binding properties of the soluble extracts indicated that allof the mutants retained at least some biological activity--see Table 2.It was concluded that mutation at Arg36 or Arg37 prevented degradationof the IGF-II by E. coli periplasmic proteases.

Example 3

Production and Purification of IGF-II (Q³⁷ Q³⁸)

The properties of the protease resistant derivative of IGF-II carried onpSD53 (Q³⁷ Q³⁸) were investigated in more detail. For larger scaleproduction, strain HW1110 carrying pSD53 was streaked on L-agar platescontaining 100 μg/ml carbenicillin and incubated overnight at 37°. Thefollowing day, a 10 ml culture of M9 minimal medium containing 100 μg/mlcarbenicillin was inoculated with a single colony and incubated at 37°overnight. This fresh overnight culture (8 ml) was used to inoculate 500ml of fresh pre-warmed M9 medium containing 100 μg/ml carbenicillin in a2 liter baffle flask. The culture was incubated at 37° C. with shakinguntil the OD₆₅₀ reached 0.4. IGF-II expression was induced at this stageby the addition of 5 ml of IPTG (0.5M) . Incubation was continued for afurther 2 hr. The cell concentration was then determined by measuringthe OD₆₅₀ of the culture. The cells were then harvested bycentrifugation at 6500 rpm for 10 min and the supernatant discarded. Thecell pellet was kept on ice until it could be processed.

The volume of solution required to carry out the osmotic shock of thecells was calculated as follows:

    Volume (ml)=OD.sub.650 X 0.0075X Broth volume (ml)

The calculated volume of cold 20% w/v sucrose, 10 mM TRIS pH 7.5 wasthen added and the cells gently resuspended. On resuspension, 0.5M EDTApH 8.0 was added to give a final concentration of 1 mM. The cellsuspension was then incubated for 10 min on ice. The cells wererecovered by centrifugation at 6500 rpm for 10 min at 4 °. Thesupernatant was discarded and the cells were rapidly resuspended in thecalculated volume of ice cold water. After a further 10 min incubationon ice, the cells were removed by centrifugation at 6500 rpm for 10 minat 4 °. The supernatant (periplasmic fraction) was decanted and used forfurther processing.

Aliquots of the periplasmic fraction, typically 40 ml, were adjustedwith 10% v/v trifluoroacetic acid (TFA) to a final concentration of 0.1%v/v TFA. After centrifugation at 3500 rpm for 10 min at 4°, the aliquotwas loaded onto a 20 ml preparative Vydac C-18 columnpre-warmed-equilibrated in 25% acetonitrile, 0.1% v/v TFA with a flowrate of 4 ml/min. After washing in the same buffer to reduce thebackground absorbance to a stable base line, the IGF-II was eluted usinga linear gradient of 25-40% v/v acetonitrile, 0.1% TFA over 45 min witha flow rate of 4 ml/min. Analysis of the active purified peak usingautomated N-terminal sequence analysis, analytical reverse phase HPLCand SDS PAGE indicates that the IGF-II (Q³⁷ Q³⁸) is >95% homogenous--seeFIG. 7A.

Example 4

Production of IGF-II and IGF-II (Q³⁷ Q³⁸) by Expression as FusionProteins in E. coli

An additional product on route for IGF-II was sought that would allowthe production of wild-type material to compare with the IGF-II cleavageresistant mutants. It was decided to produce this materialintracellularly in E. coli as a fusion protein; although this approachhas been used in the expression of small peptides, them does not haveappear to have been any realisation of the particular advantages thatflow from adapting the approach to IGF-II production. A system based onhorseradish peroxidase (HRP) was developed. HRP is an enzyme which, whenexpressed at high levels in E. coli, readily forms inclusion bodies--seeSmith et al, Journal of biological Chemistry, 265:13,335-13343 (1989).

The synthesis and expression of a synthetic gene for HRP has beendescribed (WO-A-8903424). The HRP expression plasmid pSD18 contains aNdeI-BamHI cassette cloned between the NdeI and BamHI sites of the tacexpression vector pGC517. The construction of this plasmid is describedin the above patent application. Initially, the IGF-II gene was fused tothe C-terminus of HRP by making use of an NcoI site in the HRP codingsequence.

To achieve this, pSD18 was linearised within the HRP gene with NcoI. The5' cohesive ends were removed by digestion with mung bean nuclease. Thelinearised plasmid was purified by phenol extraction and ethanolprecipitation and subjected to further digestion with BamHI. Thisprovided the vector with a blunt end within the HRP gene and a BamHIcohesive end adjacent to the transcription terminator. A fragmentcarrying IGF-II was isolated from pGC518 by linearising the plasmid withNdeI, blunt-ending by filling in the recessed ends with Klenow fragmentof DNA polymerase, and finally digesting with BamHI. TheBamHI/blunt-ended fragment encoding IGF-II was isolated byelectrophoresis and ligated into the BamHI/blunt-ended vector fragmentobtained from pGC518. The ligation products were transformed into HW1110 and the desired recombinants identified by restriction analysis ofplasmid DNA isolated from individual transformant colonies selected onL-agar plates containing 100 μg/ml carbenicillin. This procedureresulted in the construction of a gene (SEQ ID NO. 9) encoding a fusionprotein comprising the first 282 amino-acid residues of HRP fused toIGF-II via a methionine residue to allow for the release of matureIGF-II by cleavage with cyanogen bromide. The fusion protein lacks thefinal 27 amino acids of HRP. The plasmid carrying this fusion gene wasdesignated pSD24.

The fusion protein encoded by pSD24 was not ideal for the production ofIGF-II for two reasons. First, the IGF-II is only 19% of the totalfusion protein. Second, and more important, the HRP portion of themolecule contains an additional two internal methionine residues. Thisresults in the generation of additional fragments on CNBr cleavage,similar in size to IGF-II, and complicates the isolation procedure. Afusion protein was therefore engineered that carrier less residual HRPsequence. This was achieved by a similar process to that used in theconstruction of pSD24, except that an NheI site within the HRP gene wasused instead of the NcoI site. Plasmid pSD18 was linearised with NheI,the recessed ends were filled in with Klenow fragment of DNA polymeraseI, and finally digested with BamHI. The NdeI-BamHI fragment carryingIGF-II was isolated from pGC518, and the NdeI site blunt-ended, asdescribed above. The linearised pSD18 vector and the IGF-II carryingfragment were separated on low-gelling temperature gels, isolated byphenol extraction and ligated together. The ligation products weretransformed into NW1110 and the desired recombinants identified byrestriction analysis of plasmids isolated from individual transformants.The integrity of the junctional sequences was confirmed by dideoxy DNAsequencing. This procedure resulted in a gene encoding a fusion betweenthe first 53 amino acids of HRP and mature IGF-II linked by a uniquemethionine residue (SEQ ID NO. 11). This plasmid was designatedpSD28--see FIG. 8.

For production of fusion protein, an overnight culture was prepared byinoculating 10 ml of M9 minimal medium containing 100 μg/mlcarbenicillin with a single colony of HW1110/pSD28. The culture wasincubated at 37° with shaking for about 18 hr. (overnight). Theovernight culture was then used to inoculate 8×500 ml aliquots ofpre-warmed M9 minimal medium containing 100 μg/ml carbenicillin in 2liter baffle flasks. The cultures were incubated at 37° with shakinguntil the cell density measured by O.D.₆₇₀ reached 0.3-0.4. Expressionof the fusion protein was induced by the addition of 5 ml of 0.5M IPTGper flask, and the incubation continued for a further 4 hr. The cellswere then collected by centrifugation at 6,500 rpm for 10 min and thesupernatants discarded. The cell pellets were then either processedimmediately or stored at -70° until needed.

Each pellet from 500 ml of culture was then treated as follows: thecells were resuspended in 50 ml of ice-cold 50 mM TRIS pH 8.0, 50 mMNaCl, 1 mM EDTA. Lysozyme was then added to a final concentration of 1mg/ml and the cells kept on ice for 20 min. To complete the lysis, 2.75ml of sodium deoxycholate 2% w/v was added and the cells were incubatedfor 5 min at 37°. The preparation was then cooled and sonicated until nolonger viscous. The sample was then centrifuged at 15,000 rpm for 20 minto pellet the inclusion bodies. The supernatant was discarded and thepellets containing the fusion protein resuspended and washed three limesin 50 ml of a buffer comprising 0.05% Triton X100, 0.1 mM PMSF, 10 mMEDTA pH 8.0 and three times in 50 ml 3M urea, 50 mM TRIS pH 8.0. At thisstage the inclusion bodies were weighed and resuspended to 10 mg/ml informic acid 75% v/v containing sodium thiosulphate at 3% w/w ofinclusion bodies. An approximately 10 fold molar excess of cyanogenbromide (about 50 mg) was then added in a fume hood and the sampleincubated overnight at room temperature with gentle mixing (after themethod described in U.S. Pat. No. 4,451,396). The sample was then driedby rotary evaporation, washed once with an equal volume of water, driedand resuspended in 7M urea, 50 mM TRIS pH 8.0 to a final concentrationof 10 mg/ml inclusion bodies. Sodium sulphite and sodium tetrathionatewere added to final concentrations of 100 mM and 10 mM respectively andthe sample allowed to stand at room temperature to effect sulphitylationof cysteine residues.

The derivatised IGF-II was then purified by ion-exchange chromatographyon a Mono Q (Trade Mark) column, loading in 50 mM TRIS pH 8.0, 7M urea.The material was eluted with a gradient of 0-400 mM NaCl in the samebuffer over 10 column volumes. The peak containing IGF-II as determinedby gel analysis was chromatographed on a PD-10 size exclusion column in20 mM glycine, 1M urea pH 10.0 to effect buffer exchange. Cysteine wasadded to achieve an approximately 4-fold molar excess over protein,equivalent to a final concentration of about 100 μg/ml, and the mixtureallowed to stand for 16 hr. at 4 °. The reaction was quenched by theaddition of 1M HCl to a final pH of about 2.5 and the sample loaded ontoa Dynachrome FPLC cation exchange column equilibrated in 20 mM malonicacid, 1M urea pH 2.5. The IGF-II was eluted with a gradient of 0-2M NaClin the same buffer over 10 column volumes. The active, refolded IGF-IIfraction was then subjected to buffer exchange into 0.1% w/v TFA on aPD-10 column and subsequently loaded onto a Vydac analytical reversephase C-18 HPLC column and eluted with a gradient of 20-45%acetonitrile, 0.1% w/v TFA over 20 min at a flow rate of 1 ml/min.Analysis of the active purified peak using automated N-terminal sequenceanalysis, analytical reverse phase HPLC and SDS PAGE indicates that theIGF-II is >95% homogenous--see FIG. 9B.

The fusion/renaturation approach was also used to produce IGF-II(Q³⁷,Q³⁸). To construct a derivative of pSD28 carrying the Q37 and Q38mutations, a Bg/II-SalI fragment encompassing all of the HRP-IGF-IIfusion was cloned into BamHI/SalI-cut M13mp18. Single strands wereprepared and site directed mutagenesis performed with primer BB1551 asdescribed in Example 1. Phage carrying the desired mutation wereidentified by DNA sequence analysis on small scale single stranded DNApreparations from individual plaques. An isolate carrying the desiredR37Q,R38Q mutations was identified and RF DNA prepared. The cassetteencoding the mutant fusion derivative was then excised as an NdeI-BamHIfragment and cloned into NdeI/BamHI digested pGC517 to give pSD103. Thisplasmid is identical to pSD28 except for the presence of the Q37,Q38mutations.

Inclusion bodies for the HRP/IGF-II (Q³⁷,Q³⁸ derivative were obtainedand the mutant protein purified as described above for wild-type IGF-II.The biological characterisation of the refolded IGF-II, both wild-typeand mutant, is described in Example 5.

Example 5

Biological Characterisation of IGF-II (Q³⁷ Q³⁸)

The properties of the mutant IGF-II (R37Q,R38Q) were compared to thoseof wild-type material by assessing its ability to bind to the type 1 and2 IGF receptors and the insulin receptor. The mutant IGF-II was producedeither by secretion into the periplasmic space (Example 3) or byintercellular expression as a fusion protein followed by renaturation(Example 4). Two forms of wild-type IGF-II were used for comparison. Thefusion route was used to make material directly comparable to therefolded mutant derivative. In addition, recombinant IGF-II from acommercial supplier (Bachem) was used as a reference standard. Theprotein concentration of the purified homogenous material used forcharacterisation was determined by measurement of absorbance at 280 nm.

Binding of the peptides to the type 1 IGF receptor on Swiss mouse 3T3cells is shown in FIG. 10. Swiss 3T3 cells at 5×10⁴ cells/ml were seededinto 24 well plates at 5×10³ cells/well in DMEM containing 10% FCS andincubated at 37°. The cells were used for assay after 5-6 days. For thebinding assay, the growth medium was flicked out of the plate, and thecells washed twice with binding medium (serum free DMEM containing 25 mMHEPES, 1 mg/ml BSA and 2 mM glutamine). A further 400 μl of bindingmedium was added to each well along with 50 μl of sample or IGF-IIstandard (Bachem). Twofold dilutions of IGF-II were used for thestandard curve to give final concentrations in the well from 300-1.56ng/ml. Labelled IGF-I was then added (50 μl of ¹²⁵ I-IGF-I) to give afinal concentration of 1 ng/ml and a total of about 40,000 dpm per well.The plate was incubated at room temperature for 2 hr., the assay mediumremoved and the cells washed four limes in PBS containing 1 mg/ml BSAand 0.1 μM KI. The plates were blotted dry and the cells solubilised forby the addition of 750 μl 0.5M NaOH containing 0.2% w/v Triton X100.After 15 min the lysed samples were transferred to tubes and theresidual counts determined using a gamma counter.

The IGF-II (Q³⁷ Q³⁸) mutant, produced using either the secretion orrefolding route, inhibits binding of ¹²⁵ I-IGF-II with an IC₅₀ of 210ng/ml compared to 60 ng/ml and 100 ng/ml for the wild-type standard(Bachem) and wild-type refolded material respectively.

Binding to the type 2 IGF receptor was assessed on H35 (rat hepatoma)cells, see FIG. 11. Cells at 5×10⁵ cells/ml were seeded into 24 wellplates at 5×10⁴ cells/well in DMEM containing 0.5% FCS and incubated at37°. The cells were used for assay after 2-3 days. For the bindingassay, the growth medium was flicked out of the plate, and the cellswashed once with binding medium (serum free DMEM containing 25 mM HEPES,1 mg/ml BSA and 2 mM glutamine).

A further 400 μl of binding medium was added to each well along with 50μl of sample or IGF-II standard (Bachem). Twofold dilutions of IGF-IIwere used for the standard curve to give final concentrations in thewell from 300-1.56 ng/ml. Labelled IGF-II was then added (50 μl of ¹²⁵I-IGF-I) to give a final concentration of 2 ng/ml and a total of about40,000 dpm per well. The plate was incubated at room temperature for 3hr., the assay medium removed and the cells washed four times in Hanksbuffered salt solution containing 1 mg/ml BSA. The plates were blotteddry and the cells solubilised for by the addition of 750 μl 10 mM TrisHCl, 5 mM EDTA, 0.2% w/v SDS. After 15 min the lysed samples weretransferred to tubes and the residual counts determined using a gammacounter.

The mutant IGF-II (Q³⁷ Q³⁸) inhibits the binding of ¹²⁵ I-IGF-II withIC₅₀ values of 30 ng/ml (secreted) and 60 ng/ml (refolded). Thewild-type reference standard and the wild-type refolded material gavevalues of 18 ng/ml and 30 ng/ml respectively in the same assay.

Binding of the peptides to the insulin receptor, also on H35 cells, isshown in FIG. 12. The assay was performed exactly as described for thetype 2 IGF receptor except that 125I-insulin was used instead oflabelled IGF-II (Amersham). In this assay, the mutant IGF-II (Q³⁷ Q³⁸)inhibits the binding of ¹²⁵ I-insulin with IC₅₀ values of 1600 ng/ml and1300 ng/ml for secreted and refolded material respectively. In contrast,the wild-type material exhibits much higher affinity with ICs₅₀ valuesof 130 mg/ml for the standard and 160 ng/ml for the refolded material.Insulin itself has an IC₅₀ of 4.8 ng/ml in this assay.

These results show that substitution of glutamine for arginine atpositions 37 and 38 of the native IGF-II molecule decreases its affinityfor the insulin receptor by 10-fold. In contrast, the affinity of IGF-II(Q³⁷ Q³⁸) for the types 1 and 2 IGF receptor is comparable to nativeIGF-II. There is no appreciable difference in activity between materialproduced using either the secretion or HRP fusion approaches.

METHODS

All the techniques of genetic manipulation used in the manufacture ofthis gene are well known to those skilled in the art of geneticengineering. A description of most of the techniques can be found in thelaboratory manual entitled Molecular Cloning by T. Maniatis, E. F.Fritsch and J. Sambrook published by Cold Spring Harbor Laboratory, Box100, New York.

Additional and modified methodologies are detailed below.

Oligonucleotide synthesis

The oligonucleotides were synthesised by automated phosphoramiditechemistry using cyanoethyl phosphor-amidites. The methodology is nowwidely used and has been described (Beaucage, S. L. and Caruthers, M. H.Tetrahedron Letters. 24,245 (1981)).

Purification of Oligonucleotides

The oligonucleotides were de-protected and removed from the CPG supportby incubation in concentrated NH₃. Typically, 50 mg of CPG carrying 1micromole of oligo-nucleotide was de-protected by incubation for 5 hr at70° in 600 μl of concentrated NH₃. The supernatant was transferred to afresh tube and the oligomer precipitated with 3 volumes of ethanol.Following centrifugation the pellet was dried and resuspended in 1 ml ofwater. The concentration of crude oligomer was then determined bymeasuring the absorbance at 260 nm.

For gel purification 10 absorbance units of the crude oligonucleotidewere dried down and resuspended in 15 μl of marker dye (90% de-ionisedformamide, 10 mM tris, 10 mM borate, 1 mM EDTA, 0.1% bromophenol blue).The samples were heated at 90° for 1 minute and then loaded onto a 1.2mm thick denaturing polyacrylamide gel with 1.6 mm wide slots. The gelwas prepared from a stock of 15% acrylamide, 0.6% bisacrylamide and 7Murea in 1 XTBE and was polymerised with 0.1% ammonium persulphate and0.025% TEMED. The gel was pre-warmed-run for 1 hr. The samples were runat 1500 V for 4-5 hr. The bands were visualised by UV shadowing andthose corresponding to the full length product cut out and transferredto micro-testtubes. The oligomers were eluted from the gel slice bysoaking in AGEB (0.5M ammonium acetate, 0.01 M magnesium acetate and0.1% SDS) overnight. The AGEB buffer was then transferred to fresh tubesand the oligomer precipitated with three volumes of ethanol at -70° for15 min. The precipitate was collected by centrifugation in an Eppendorfmicrofuge for 10 min, the pellet washed in 80% ethanol, the purifiedoligomer dried, redissolved in 1 ml of water and finally filteredthrough a 0.45 μm microfilter. The concentration of purified product wasmeasured by determining its absorbance at 260 nm.

Kinasing of oligomers

250 pmole of oligomer was dried down and resuspended in 20 μl kinasebuffer (70 mM Tris pH7.6, 10 mM MgCl₂, 1 mM ATP, 0.2 mM spermidine, 0.5mM dithiothreitol). 10 u of T4 polynucleotide kinase was added and themixture incubated at 37° for 30 min. The kinase was then inactivated byhealing at 85° for 15 min.

Isolation of DNA from agarose gels

The volume of the gel slice was estimated from its weight and thenmelted by incubation at 65° for 10 min. The volume of the slice was thenmade up to 400 μl with TE (10 mM Tris pH 8.0, 1 mM EDTA) and Na acetateadded to a final concentration of 0.3M. 10 μg of yeast tRNA was alsoadded as a carrier. The DNA was then subjected to three rounds ofextraction with equal volumes of TE equilibrated phenol followed bythree extractions with ether that had been saturated with water. The DNAwas precipitated with 2 volumes of ethanol, centrifuged for 10 min in aMicrofuge, the pellet washed in 70% ethanol and finally dried down.

Dideoxy sequencing

The protocol used was essentially as has been described (Biggin, M. D.,Gibson, T. J., Hong, G.F.P.N.A.S. 80 3963-3965 (1983)).

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 12                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 67 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Cleavage-site                                                   (B) LOCATION: 37..38                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AlaTyrArgProSerGluThrLeuCysGlyGlyGluLeuValAspThr                              151015                                                                        LeuGlnPheValCysGlyAspArgGlyPheTyrPheSerArgProAla                              202530                                                                        SerArgValSerArgArgSerArgGlyIleValGluGluCysCysPhe                              354045                                                                        ArgSerCysAspLeuAlaLeuLeuGluThrTyrCysAlaThrProAla                              505560                                                                        LysSerGlu                                                                     65                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 67 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: HOMO SAPIENS                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 37..38                                                          (D) OTHER INFORMATION: /note= "Protease resistant mutant                      form of IGF- II with Arg-37 modified to Gln and                               Arg-38 modified to Gln. Resistance to cleavage by                             the E. coli periplasmic protease and reduced affinity                         for the insulin receptor                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AlaTyrArgProSerGluThrLeuCysGlyGlyGluLeuValAspThr                              151015                                                                        LeuGlnPheValCysGlyAspArgGlyPheTyrPheSerArgProAla                              202530                                                                        SerArgValSerGlnGlnSerArgGlyIleValGluGluCysCysPhe                              354045                                                                        ArgSerCysAspLeuAlaLeuLeuGluThrTyrCysAlaThrProAla                              505560                                                                        LysSerGlu                                                                     65                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 221 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 9..212                                                          (D) OTHER INFORMATION: /codon.sub.-- start= 9                                 /function= "Synthetic gene for mature human                                   IGF-ii"                                                                       /product= "Synthetic DNA"                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /function="SphI site"                                  /number= 1                                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 6..12                                                           (D) OTHER INFORMATION: /function="NdeI site"                                  /number= 2                                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 216..221                                                        (D) OTHER INFORMATION: /function="BamHI cleavage site"                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GCATGCATATGGCATACCGCCCGAGCGAGACCCTGTGCGGTGGCGAGCTC50                          MetAlaTyrArgProSerGluThrLeuCysGlyGlyGluLeu                                    1510                                                                          GTAGACACTCTGCAGTTCGTTTGTGGTGACCGTGGCTTCTACTTCTCT98                            ValAspThrLeuGlnPheValCysGlyAspArgGlyPheTyrPheSer                              15202530                                                                      CGTCCTGCTAGCCGTGTATCTCGCCGTTCTAGAGGCATCGTTGAAGAG146                           ArgProAlaSerArgValSerArgArgSerArgGlyIleValGluGlu                              354045                                                                        TGCTGTTTCCGCAGCTGTGATCTGGCACTGCTCGAAACTTACTGCGCA194                           CysCysPheArgSerCysAspLeuAlaLeuLeuGluThrTyrCysAla                              505560                                                                        ACTCCAGCAAAATCCGAATAAGGATCC221                                                ThrProAlaLysSerGlu                                                            65                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 68 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetAlaTyrArgProSerGluThrLeuCysGlyGlyGluLeuValAsp                              151015                                                                        ThrLeuGlnPheValCysGlyAspArgGlyPheTyrPheSerArgPro                              202530                                                                        AlaSerArgValSerArgArgSerArgGlyIleValGluGluCysCys                              354045                                                                        PheArgSerCysAspLeuAlaLeuLeuGluThrTyrCysAlaThrPro                              505560                                                                        AlaLysSerGlu                                                                  65                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 102 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 2..102                                                          (D) OTHER INFORMATION: /codon.sub.-- start= 2                                 /function= "Synthetic ompA leader sequence"                                   /product= "Synthetic DNA"                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /function="NdeI cleavage site"                         (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 100..102                                                        (D) OTHER INFORMATION: /function="SacI cleavage site"                         (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 2..65                                                           (D) OTHER INFORMATION: /function="ompA leader sequence"                       (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 66..102                                                         (D) OTHER INFORMATION: /function="IGF-II gene (part)"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTC46                              MetLysLysThrAlaIleAlaIleAlaValAlaLeuAlaGlyPhe                                 151015                                                                        GCGACCGTAGCGCAGGCCGCATACCGCCCGAGCGAGACCCTGTGCGGT94                            AlaThrValAlaGlnAlaAlaTyrArgProSerGluThrLeuCysGly                              202530                                                                        GGCGAGCT102                                                                   GlyGluLeu                                                                     (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetLysLysThrAlaIleAlaIleAlaValAlaLeuAlaGlyPheAla                              151015                                                                        ThrValAlaGlnAlaAlaTyrArgProSerGluThrLeuCysGlyGly                              202530                                                                        GluLeu                                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..23                                                           (D) OTHER INFORMATION: /function="directs substitution of                     Gln for Arg at positions 37 and 38"                                           /product= "IGF-II mutagenesis primer"                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CCTCTAGATTGCTGAGATACACG23                                                     (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..24                                                           (D) OTHER INFORMATION: /function="directs random                              substitution at Arg-37 and Arg-38"                                            /product= "IGF-II mutagenesis primer"                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GCCTCTAGANNNNNNAGATACACG24                                                    (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1072 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 11..1057                                                        (D) OTHER INFORMATION: /codon.sub.-- start= 11                                /function= "Gene for IGF-II/HRP fusion protein"                               /product= "Synthetic DNA"                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /function="HinDIII cleavage site"                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1067..1072                                                      (D) OTHER INFORMATION: /function="EcoRI cleavage site"                        (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 11..856                                                         (D) OTHER INFORMATION: /function="HRP coding sequence                         (fragment)"                                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 857..1057                                                       (D) OTHER INFORMATION: /function="IGF-II coding sequence"                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       AAGCTTAACCATGCAGTTAACCCCTACATTCTACGACAATAGCTGTCCC49                           MetGlnLeuThrProThrPheTyrAspAsnSerCysPro                                       1510                                                                          AACGTGTCCAACATCGTTCGCGACACAATCGTCAACGAGCTCAGATCC97                            AsnValSerAsnIleValArgAspThrIleValAsnGluLeuArgSer                              152025                                                                        GATCCCAGGATCGCTGCTTCAATATTACGTCTGCACTTCCATGACTGC145                           AspProArgIleAlaAlaSerIleLeuArgLeuHisPheHisAspCys                              30354045                                                                      TTCGTGAATGGTTGCGACGCTAGCATATTACTGGACAACACCACCAGT193                           PheValAsnGlyCysAspAlaSerIleLeuLeuAspAsnThrThrSer                              505560                                                                        TTCCGCACTGAAAAGGATGCATTCGGGAACGCTAACAGCGCCAGGGGC241                           PheArgThrGluLysAspAlaPheGlyAsnAlaAsnSerAlaArgGly                              657075                                                                        TTTCCAGTGATCGATCGCATGAAGGCTGCCGTTGAGTCAGCATGCCCA289                           PheProValIleAspArgMetLysAlaAlaValGluSerAlaCysPro                              808590                                                                        CGAACAGTCAGTTGTGCAGACCTGCTGACTATAGCTGCGCAACAGAGC337                           ArgThrValSerCysAlaAspLeuLeuThrIleAlaAlaGlnGlnSer                              95100105                                                                      GTGACTCTTGCAGGCGGACCGTCCTGGAGAGTGCCGCTCGGTCGACGT385                           ValThrLeuAlaGlyGlyProSerTrpArgValProLeuGlyArgArg                              110115120125                                                                  GACTCCCTACAGGCATTCCTAGATCTGGCCAACGCCAACTTGCCTGCT433                           AspSerLeuGlnAlaPheLeuAspLeuAlaAsnAlaAsnLeuProAla                              130135140                                                                     CCATTCTTCACCCTGCCCCAGCTGAAGGATAGCTTTAGAAACGTGGGT481                           ProPhePheThrLeuProGlnLeuLysAspSerPheArgAsnValGly                              145150155                                                                     CTGAATCGCTCGAGTGACCTTGTGGCTCTGTCCGGAGGACACACATTT529                           LeuAsnArgSerSerAspLeuValAlaLeuSerGlyGlyHisThrPhe                              160165170                                                                     GGAAAGAACCAGTGTAGGTTCATCATGGATAGGCTCTACAATTTCAGC577                           GlyLysAsnGlnCysArgPheIleMetAspArgLeuTyrAsnPheSer                              175180185                                                                     AACACTGGGTTACCTGACCCCACGCTGAACACTACGTATCTCCAGACA625                           AsnThrGlyLeuProAspProThrLeuAsnThrThrTyrLeuGlnThr                              190195200205                                                                  CTGAGAGGCTTGTGCCCACTGAATGGCAACCTCAGTGCACTAGTGGAC673                           LeuArgGlyLeuCysProLeuAsnGlyAsnLeuSerAlaLeuValAsp                              210215220                                                                     TTTGATCTGCGGACCCCAACCATCTTCGATAACAAGTACTATGTGAAT721                           PheAspLeuArgThrProThrIlePheAspAsnLysTyrTyrValAsn                              225230235                                                                     CTAGAGGAGCAGAAAGGCCTGATACAGAGTGATCAAGAACTGTTTAGC769                           LeuGluGluGlnLysGlyLeuIleGlnSerAspGlnGluLeuPheSer                              240245250                                                                     AGTCCAAACGCCACTGACACCATCCCACTGGTGAGAAGTTTTGCTAAC817                           SerProAsnAlaThrAspThrIleProLeuValArgSerPheAlaAsn                              255260265                                                                     TCTACTCAAACCTTCTTTAACGCCTTCGTGGAAGCTATGGCATACCGC865                           SerThrGlnThrPhePheAsnAlaPheValGluAlaMetAlaTyrArg                              270275280285                                                                  CCGAGCGAGACCCTGTGCGGTGGCGAGCTCGTAGACACTCTGCAGTTC913                           ProSerGluThrLeuCysGlyGlyGluLeuValAspThrLeuGlnPhe                              290295300                                                                     GTTTGTGGTGACCGTGGCTTCTACTTCTCTCGTCCTGCTAGCCGTGTA961                           ValCysGlyAspArgGlyPheTyrPheSerArgProAlaSerArgVal                              305310315                                                                     TCTCGCCGTTCTAGAGGCATCGTTGAAGAGTGCTGTTTCCGCAGCTGT1009                          SerArgArgSerArgGlyIleValGluGluCysCysPheArgSerCys                              320325330                                                                     GATCTGGCACTGCTCGAAACTTACTGCGCAACTCCAGCAAAATCCGAA1057                          AspLeuAlaLeuLeuGluThrTyrCysAlaThrProAlaLysSerGlu                              335340345                                                                     TAAGGATCCGAATTC1072                                                           (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 349 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      MetGlnLeuThrProThrPheTyrAspAsnSerCysProAsnValSer                              151015                                                                        AsnIleValArgAspThrIleValAsnGluLeuArgSerAspProArg                              202530                                                                        IleAlaAlaSerIleLeuArgLeuHisPheHisAspCysPheValAsn                              354045                                                                        GlyCysAspAlaSerIleLeuLeuAspAsnThrThrSerPheArgThr                              505560                                                                        GluLysAspAlaPheGlyAsnAlaAsnSerAlaArgGlyPheProVal                              65707580                                                                      IleAspArgMetLysAlaAlaValGluSerAlaCysProArgThrVal                              859095                                                                        SerCysAlaAspLeuLeuThrIleAlaAlaGlnGlnSerValThrLeu                              100105110                                                                     AlaGlyGlyProSerTrpArgValProLeuGlyArgArgAspSerLeu                              115120125                                                                     GlnAlaPheLeuAspLeuAlaAsnAlaAsnLeuProAlaProPhePhe                              130135140                                                                     ThrLeuProGlnLeuLysAspSerPheArgAsnValGlyLeuAsnArg                              145150155160                                                                  SerSerAspLeuValAlaLeuSerGlyGlyHisThrPheGlyLysAsn                              165170175                                                                     GlnCysArgPheIleMetAspArgLeuTyrAsnPheSerAsnThrGly                              180185190                                                                     LeuProAspProThrLeuAsnThrThrTyrLeuGlnThrLeuArgGly                              195200205                                                                     LeuCysProLeuAsnGlyAsnLeuSerAlaLeuValAspPheAspLeu                              210215220                                                                     ArgThrProThrIlePheAspAsnLysTyrTyrValAsnLeuGluGlu                              225230235240                                                                  GlnLysGlyLeuIleGlnSerAspGlnGluLeuPheSerSerProAsn                              245250255                                                                     AlaThrAspThrIleProLeuValArgSerPheAlaAsnSerThrGln                              260265270                                                                     ThrPhePheAsnAlaPheValGluAlaMetAlaTyrArgProSerGlu                              275280285                                                                     ThrLeuCysGlyGlyGluLeuValAspThrLeuGlnPheValCysGly                              290295300                                                                     AspArgGlyPheTyrPheSerArgProAlaSerArgValSerArgArg                              305310315320                                                                  SerArgGlyIleValGluGluCysCysPheArgSerCysAspLeuAla                              325330335                                                                     LeuLeuGluThrTyrCysAlaThrProAlaLysSerGlu                                       340345                                                                        (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 388 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 11..373                                                         (D) OTHER INFORMATION: /codon.sub.-- start= 11                                /function= "Gene for IGF-II/HRP fusion protein"                               /product= "Synthetic DNA"                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /function="HindIII cleavage site"                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 383..388                                                        (D) OTHER INFORMATION: /function="EcoRI cleavage site"                        (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 11..172                                                         (D) OTHER INFORMATION: /function="HRP coding sequence"                        (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 173..373                                                        (D) OTHER INFORMATION: /function="IGF-II coding sequence"                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      AAGCTTAACCATGCAGTTAACCCCTACATTCTACGACAATAGCTGTCCC49                           MetGlnLeuThrProThrPheTyrAspAsnSerCysPro                                       1510                                                                          AACGTGTCCAACATCGTTCGCGACACAATCGTCAACGAGCTCAGATCC97                            AsnValSerAsnIleValArgAspThrIleValAsnGluLeuArgSer                              152025                                                                        GATCCCAGGATCGCTGCTTCAATATTACGTCTGCACTTCCATGACTGC145                           AspProArgIleAlaAlaSerIleLeuArgLeuHisPheHisAspCys                              30354045                                                                      TTCGTGAATGGTTGCGACGCTAGTATGGCATACCGCCCGAGCGAGACC193                           PheValAsnGlyCysAspAlaSerMetAlaTyrArgProSerGluThr                              505560                                                                        CTGTGCGGTGGCGAGCTCGTAGACACTCTGCAGTTCGTTTGTGGTGAC241                           LeuCysGlyGlyGluLeuValAspThrLeuGlnPheValCysGlyAsp                              657075                                                                        CGTGGCTTCTACTTCTCTCGTCCTGCTAGCCGTGTATCTCGCCGTTCT289                           ArgGlyPheTyrPheSerArgProAlaSerArgValSerArgArgSer                              808590                                                                        AGAGGCATCGTTGAAGAGTGCTGTTTCCGCAGCTGTGATCTGGCACTG337                           ArgGlyIleValGluGluCysCysPheArgSerCysAspLeuAlaLeu                              95100105                                                                      CTCGAAACTTACTGCGCAACTCCAGCAAAATCCGAATAAGGATCCG383                             LeuGluThrTyrCysAlaThrProAlaLysSerGlu                                          110115120                                                                     AATTC388                                                                      (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 121 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      MetGlnLeuThrProThrPheTyrAspAsnSerCysProAsnValSer                              151015                                                                        AsnIleValArgAspThrIleValAsnGluLeuArgSerAspProArg                              202530                                                                        IleAlaAlaSerIleLeuArgLeuHisPheHisAspCysPheValAsn                              354045                                                                        GlyCysAspAlaSerMetAlaTyrArgProSerGluThrLeuCysGly                              505560                                                                        GlyGluLeuValAspThrLeuGlnPheValCysGlyAspArgGlyPhe                              65707580                                                                      TyrPheSerArgProAlaSerArgValSerArgArgSerArgGlyIle                              859095                                                                        ValGluGluCysCysPheArgSerCysAspLeuAlaLeuLeuGluThr                              100105110                                                                     TyrCysAlaThrProAlaLysSerGlu                                                   115120                                                                        __________________________________________________________________________

We claim:
 1. An insulin-like Growth Factor II (IGF-II) analogue in whichat least one of R37 and R38, wherein R37 represents the natural arginineresidue at position 37 and R38 represents the natural arginine residueat position 38 of SEQ. ID NO. 1, is replaced with another amino acidresidue.
 2. An analogue as claimed in claim 1, wherein both R37 and R38are replaced.
 3. An analogue as claimed in claim 1, wherein the or eachreplacement amino acid residue is non-basic.
 4. An analogue as claimedin claim 3, wherein the or each replacement amino acid residue is aneutral residue other than Pro.
 5. An analogue as claimed in claim 4,wherein the or each replacement amino acid residue is an amide residue.6. An analogue as claimed in claim 4, wherein the or each replacementamino acid residue is Gln.
 7. IGF-II (Arg37Gln Arg38Gln).
 8. An IGF-IIanalogue as claimed in claim 1 for use in medicine.
 9. A pharmaceuticalcomposition comprising an IGF-II analogue as claimed in claim 1 and apharmaceutically acceptable carrier therefore.